THICK AND THIN SMEAR PREPARATION

FOR
MALARIA MICROSCOPY
Advanced Quantitative Microscopy Training Workshop
Jakarta, October 2019
 THE BURDENS of MALARIA
• 216 million malaria cases/year in the world
194 million malaria in Africa in 2017
• 445 000 deaths/year (mostly children under 5 y.o.)
407 000 death in Africa in 2017
• The costs of malaria are also enormous when measured
in economic terms.
• Malaria has played a significant role in the economic
performance of endemic countries.
Challenges in Malaria Research
ALL OF MALARIA RESEARCHS NEED
ACCURATE MALARIA DIAGNOSIS
1 • Clinical Diagnosis

2 • Malaria Blood Smear

3 • Fluorescent microscopy

4 • Antigen Detection

5 • Serology

6 • PCR
MALARIA BLOODSMEAR
• Remains the gold standard for diagnosis and clinical trials
Giemsa stain
distinguishes between species and life cycle stages
parasitemia is quantifiable

• Threshold of detection
thin film: 100 parasites/ul
thick film: 5 -20 parasites/ul

• Requirements: equipment, training, reagents, supervision

• Simple, inexpensive yet labor-intensive
• Accuracy depends on laboratorian skill
MPETENT MICROSCOPIST IS REQUIRED IN MALARIA VACCINE T

Competent Microscopist: A Microscopist who passes certification

testing for preparation, staining and reading of thick blood smears
for malaria diagnosis.

S/he must be able to:

1. Prepare thick and thin blood smears according to the clinical
trial SOP;
2. Be able to detect parasites and densities ≥9 parasites/uL in
>90% of samples;
3. Read thick blood smears with no false positives.
THICK AND THIN BLOODSMEAR
The World Health Organization recommends counting
about 0.5 microliters of blood in clinical trials of
malaria vaccines (Consensus SOP for Malaria
Microscopy in the Context of Clinical Challenge Trials).

This volume is approximately the same as the volume

counted in previous clinical trials, which was the result
of counting 200 high-power fields of a circular thick
smear with a diameter of 1.0-1.6 centimeters.
 THICK AND THIN
BLOODSMEAR
• THICK FILM • THIN FILM
– lysed RBCs – fixed RBCs, single layer
– larger volume – smaller volume
– 0.25 μl blood/100 fields – 0.005 μl blood/100
– blood elements are not fields
concentrated
– good species
– good screening test
differentiation
– positive or negative
– requires more time to
– parasite density
read
– more difficult to
diagnose species – low density infections
can be missed
THICK AND THIN BLOODSMEAR
FOR
PFSPZ MALARIA
Better screening test: VACCINE TRIALS
positive or negative
Exact volume TBS: 10uL
to measure parasite density
Size: 1cm x 2cm
blood elements are not concentrated

Thin smear to allows improved species differentiation

compared to thick blood smear technique
Prepare smears as soon as possible after
collecting venous blood to avoid
 Changes in parasite morphology
 Staining characteristics
EQUIPMENTS:
• Pre-cleaned slide glass
• Slide template (size 10mm x 20mm)
• Pipettor 20uL with pipet tip
• Slide warmer
• Timer
• Staining equipments
• Slide rack
Thick and thin smear preparation

Place 10 L blood in the rectangle

and 2 L in the circle for

making thin smear.
Thin smear preparation

Slide label

First pull this direction

Drop of 2ul for thin smear
HOW TO MAKE THE THICK
BLOODSMEAR?
• Thick Smear Preparation

• Giemsa Staining Preparation

• Staining Procedure

• Reading / Recording Result

 Drying the blood smears:
• Allow the slides to air-dry until the blood in the
tick smear appears dry for maximum 60 minutes

• Further dry slides on a slide warmer at set to

37°C for 25 -35 minutes .

• Drying the blood smears at temperatures above

40˚C can result in a greenish tinge to the staining,
making slides more difficult to read and reducing
sensitivity.
 Fix the thin blood smear.
• Fix the thin blood smear by dipping the thin blood smear portion
of the slide in absolute methanol for 1-2 seconds and then letting
the slide air dry for a minimum of 30 seconds.

• Do not allow the methanol to flow onto the thick film.

• Dry the thin blood smear at an acute angle, with the blood smear
side of the slide facing up and the thin blood smear end of the
slide at the lower end (closest) to the rack/table.

• The thick blood smear must not be fixed.

• The blood smear is routinely stained within 6 hours of

preparation and should be stained within 24 hours.
 Reagent Preparation – pH 7.2 Buffer Solution
• Prepare the pH 7.2 buffer solution using the buffer tablet
according to the manufacturer’s instructions.
• Check the pH of the buffer solution and remake if
necessary.
• Log the preparation of the buffer solution in Form F816-
01 every time you make a fresh lot.
• Buffer can be alliquoted in 50-mL tubes, 48 mL per tube
in preparation of working solution
• Label with : “Giemsa Buffer, pH 7,2”m date of
preparation, and date of expiration (after 4 weeks of
preparation)
Reagent Preparation - Giemsa 4% Stock Solution
• The 4% or 10% working dilution Giemsa stain is prepared fresh from a
commercial 40% stock solution.
• Do not move, shake, swirl, or invert the stock bottle before taking stain
from it. There is sediment that accumulates at the bottom of a Giemsa stock
bottle, and care should be taken to avoid putting it on the slides.
• Never pipette the stock Giemsa from the bottom of the bottle to avoid the
sediment.
• Use a freshly-made Giemsa solution for each batch of 15 slides .
• Add 48 mL of buffered water to a 50 mL conical tube (tubes of buffer can be
prepared ahead of time).
• Using a new pipette add 2 mL of Giemsa stock solution to the conical tube.
• Mix by inverting 5 times.
• The 4% working Giemsa solution expires in 15 minutes.
• Time of staining 4% Giemsa solution is 45+5 minutes.
Time of staning 10% Giemsa solution is 10+2 minutes (this is generally used
for symptomatic individuals as requested urgently by physician)
THANK YOU...
ASSIGNMENT 1:

1. MAKE 10 THICK AND THIN FILM

AS DESCRIBED IN THE SOP
2. PREPARE GIEMSA DILUTION (4%)
3. GIEMSA STAINING (4% IN 45+5)